Many researches have been performed to clarify the pathogenesis of cholesteatomatous otitis media. They include the ultrastructural study of the cholesteatoma by electron microscopy and the role of keratinocyte, Langerhans’ cell, mast cell and various kinds of cytokine in pathogenesis of this disease. These studies have limitation and been done at level of the tissue. It has been known that the cell culture of cholesteatoma epithelium was difficult due to frequent contamination on culture, lack of a specimen obtained and difficulty in establishment of optimal condition on culture. Recent advances in culture methods can contribute to the study of normal physiology or pathogenesis at level of the cell. So the authors have an interest in study of individual cell biology of cholesteatoma and established cell line from cholesteatoma epithelium. The specimen was obtained on ear surgery and the cells were separated by 3T3L1 cell feeder layer technique. The availabilty of the cell line would provide a potential tool in future cholesteatoma research.